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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified. This exon, here named exon 0, contained the entire 5' untranslated region and the N-terminal signal sequence of the polypeptide. Exon 0 was isolated from a 1.6-kilobase (kb) EcoRI-HindIII fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain approximately 940 base pairs (bp) of 5'-flanking sequence, exon 0, which was approximately 245 bp in length, and approximately 400 bp of the following intron 0. By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays. Both primer extensions and nuclease S1 protection assays of postnatal day 7 rat brain RNA identified seven initiation sites within a single 10-bp region at positions -195 to -186 relative to the translation start site. An additional minor site was found at position -329. In the immediate 5' region, no consensus TATA or CCAAT sequences were found. Potential regulatory elements within this region include Sp1 consensus binding sites and also a 178-bp homopurine-homopyrimidine sequence containing several mirror repeats. NCAM has multiple transcripts which are regulated in a developmental and tissue-specific fashion. To determine whether these transcripts are initiated at the same sites, transcription initiation sites were analyzed in postnatal day 7 and adult rat brain and also in cultured cell lines of neuronal, glial, and muscle phenotypes. These tissues and cells exhibited distinct NCAM transcript populations in Northern (RNA) dot blot analysis. In all cases similar transcription start sites were found, suggesting that all major NCAM transcripts have similar or identical initiation sites. These results provide essential information to begin analysis of NCAM regulation in different tissues and during development.
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PMID:Transcription initiation sites and structural organization of the extreme 5' region of the rat neural cell adhesion molecule gene. 169 9

In the adult mammalian ovary morphogenesis and differentiation processes are under hormonal control and, thus, occur in a highly regulated way during the sexual cycle. Cell-cell interactions, such as cell adhesion and cell separation, are crucial during these events. Here we show that the ovarian endocrine cells, which are prototypes of steroid-producing cells, express neural cell adhesion molecules (NCAMs). The combined use of in situ hybridization histochemistry, immunocytochemistry at the light and electron microscope levels, S1 nuclease protection assays, and Western blotting revealed that in the ovary of the adult rat during the estrus cycle and pregnancy, NCAM mRNA and the 140-kDa isoform of this protein are expressed mainly in granulosa cells of growing preantral and antral follicles and in corpora lutea. Since the granulosa cells lining the forming antrum and the antral fluid were strongly immunoreactive, a role for NCAM in the formation of the follicular antrum is proposed. The expression of NCAM was also associated with luteal cells of the active corpus luteum, indicating a role for NCAM in the morphogenesis of this endocrine compartment. Moreover, thecal cells of large follicles and hypertrophic thecal cells of atretic follicles expressed NCAM, as did interstitial cells, which are derived from thecal cells of atretic follicles. We propose that the adhesion molecule, NCAM, is an important factor involved in the recognition and intercellular interaction of ovarian endocrine cells and, thus, participates in the regulation of the cyclic remodeling processes of the ovarian endocrine compartments.
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PMID:Expression of the neural cell adhesion molecule in endocrine cells of the ovary. 185 76

The neural cell adhesion molecule (NCAM) is thought to be involved in several important events during CNS vertebrate development. This study provides additional information concerning the biochemical determination and anatomical localization of NCAM transcripts. Using S1 nuclease protection assays (S1-NPAs), NCAM transcripts in brain appear highest at birth, with NCAM messenger levels reduced some 20-fold by adulthood. By use of in situ hybridization, NCAM mRNA is demonstrated to be developmentally regulated in the cerebellum and hippocampus. The in situ hybridization findings, in addition to providing results to compare with past studies of NCAM immunolocalization, reveal that NCAM expression in dentate gyrus granule cells and cerebellar Purkinje cells is correlated with the final stages of axonal growth, e.g., synaptic stabilization. In situ hybridization demonstrates a developmental outside-to-inside gradient of NCAM transcripts in the dentate gyrus. Neurological mutant mice, reeler and stagger, provide evidence that NCAM expression is normal in the brain regions investigated, and does not correlate with the developmental perturbations present in these strains.
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PMID:NCAM gene expression during the development of cerebellum and dentate gyrus in the mouse. 233 83

Myotube mRNA isoforms of the neural cell adhesion molecule (N-CAM) contain a novel sequence block termed muscle-specific domain 1 (MSD1), which is inserted within the extracellular coding region. Here, we report a characterization of the genomic organization of MSD1 and its pattern of expression within cellular N-CAM RNA and polypeptide species. S1 nuclease protection analyses and sequence analysis of an N-CAM human genomic clone containing MSD1 sequences indicated that MSD1 is comprised of three discrete exons of 15, 48, and 42 bp, designated MSD1a, MSD1b, and MSD1c, respectively. Although the MSD1a exon was present in a small proportion of mRNAs from both brain and muscle cells, the entire MSD1 sequence occurred predominantly in mRNAs from differentiated myotube cells. In addition, antiserum raised to a synthetic, MSD1b-encoded peptide sequence was found to stain the cell surface of human skeletal myotubes in culture, whereas myoblasts, fibroblasts, and neural cells were negative. MSD1a, MSD1b, and MSD1c sequences thus arise collectively in N-CAM mRNA and polypeptide isoforms as a result of muscle tissue-specific and developmentally regulated alternative mRNA splicing events. In addition, the occurrence of brain and muscle mRNAs containing only MSD1a indicate that alternative splicing may occur within the MSD region itself to generate further diversity.
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PMID:Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domain structure in skeletal muscle and brain. 254 26

The murine neural cell adhesion molecule (NCAM) is known to exist in three isoforms of different size, NCAM-180, -140 and -120 coded for by four transcripts of 6.9, 6.1, 4.8 and 2.7 kb in length. Since the differences between these isoforms are due to alternative splicing in the coding region for the transmembrane and cytoplasmic domains, the extracellular, N-terminal portion of NCAM seemed to be shared by all three protein forms. Here we report that the coding region for N-terminal domains of NCAM also contains at least two sites of alternative splicing, termed alpha and pi. Short additional sequences of 3, 18 and 30 nt in length can be introduced at these sites, which are located in the membrane-proximal 'stem' between the Ig-like domains and the membrane attachment site and within the Ig-like domain IV, respectively. Proof for at least eight different mRNAs has been found by sequencing and S1 nuclease protection assays of selected independent cDNA clones, and Northern blot analyses. If most combination of the splice patterns identified so far in mouse brain occurred, 24 different mRNAs could be generated coding for 18 different proteins. The shortest extra-sequence found inserted at splice site alpha consisted only of the trinucleotide AAG, raising questions about the mechanism of this particular insertion.
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PMID:Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. 272 86

The neural cell adhesion molecule (NCAM) exists in several isoforms which are selectively expressed by different cell types and at different stages of development. In the mouse, three proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished that are encoded by 4-5 different mRNAs. Here we report the full amino acid sequence of a NCAM protein inferred from the sequences of overlapping cDNA clones. The 706-residue polypeptide contains, towards its N-terminus, 5 domains that share structural homology with members of the immunoglobulin supergene family. The sequence does not encode a typical membrane-spanning segment, but ends with 24 uncharged amino acids followed by two stop codons. This fact, together with size considerations, make it highly likely that our sequence represents NCAM-120, which lacks transmembrane or cytoplasmic domains and is attached to the membrane by phospholipid. Probes from the 5' region detect all four NCAM gene transcripts present in mouse brain consistent with the notion that the extracellular domains are common to most NCAM forms. However, a 3' probe corresponding to the hydrophobic tail and non-coding region hybridizes specifically with the smallest mRNA species. S1 nuclease protection experiments indicate that this region is encoded by exon(s) spliced out from the other mRNAs. Furthermore, our clones that are highly homologous to a published chicken NCAM sequence which codes for putative transmembrane and cytoplasmic domains elsewhere, diverge from it at the presumptive splice junction. It appears thus that alternate use of exons determines whether NCAM proteins with membrane-spanning domains are synthesized.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and nucleotide sequence of mouse NCAM cDNA that codes for a Mr 79,000 polypeptide without a membrane-spanning region. 359 63

The neural cell adhesion molecule (NCAM) exists in at least three different isoforms. In the mouse, NCAM proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished. These are encoded by 4 to 5 different transcripts. Here we report the full amino acid sequence of an isoform which most likely represents NCAM-140. The N-terminal extracellular portion of the 829-residue polypeptide appears to be identical to all three NCAM proteins. The Mr of 91,276 is considerably smaller than the estimate based on SDS-gel electrophoresis. The 147 C-terminal residues are distinct from NCAM-120 and contain the putative transmembrane and cytoplasmic domains. The transcript encoding NCAM-140 contains almost 3.2 kb non-coding sequence with a canonical polyadenylation signal. While the 5' sequences of NCAM-140 hybridize with all NCAM mRNAs, the 3' probes recognize only the two larger transcripts of 7.4 and 6.7 kb. From S1 nuclease protection analyses and hybridization studies of several NCAM cDNA clones with genomic NCAM sequences one can conclude that the different NCAM transcripts are generated by alternative splicing. In addition to the two alternative splice sites in the sequence encoding the extracellular domains, a third one can be predicted approximately 320 nt downstream of the start of the NCAM-140-specific sequence portion. This finding is in agreement with the existence of an extra exon in the chicken NCAM-180. Comparison between mouse and chicken NCAM amino acid sequences revealed the highest homology in the second and fifth Ig-like domains and in the cytoplasmic parts suggesting that these regions serve highly conserved functions.
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PMID:Analysis of cDNA clones that code for the transmembrane forms of the mouse neural cell adhesion molecule (NCAM) and are generated by alternative RNA splicing. 368 67

The adhesive properties of neural cell adhesion molecules (NCAMs) can be modified by alternative splicing of the primary transcript or posttranslational modifications. In the present study, we describe distinct forms of alternative splicing and posttranslational modification of the extracellular domain of NCAM of various endocrine tissues and derived tumor cells of the rat. Using an antiserum detecting the immunoglobulin-like domains of NCAM as well as a monoclonal antibody recognizing the NCAM-specific polysialic acid (PSA), we observed a similar staining pattern in adrenals, pituitary, and neoplastic endocrine cells. In endocrine tumor cells [pheochromocytoma (PC12), insulinoma (RINA2), and pituitary tumor cells (GH3)], NCAM immunoreactivity was most intense at contact sites between the cells. The immunocytochemical data were substantiated by results of in situ hybridization histochemistry. Specifically, higher levels of NCAM mRNA were detected in the adrenal cortex than in the medulla. In the pituitary, NCAM mRNA was more abundant in the anterior and intermediate lobes than in the neural lobe. The sequence of NCAM mRNAs in endocrine cells was analyzed by polymerase chain reaction and S1 nuclease protection assays. We found that major exons 4-13 of the NCAM mRNA in endocrine tissues and related tumor cell lines were homologous to those in the brain. However, PC12, RINA2, and GH3 tumor cells; normal rat pituitaries; and adrenals contained different amounts of NCAM mRNA with an alternative extra exon, termed VASE (also called pi in mouse) between constitutive exons 7 and 8. In addition, in pituitaries, we detected an alternative exon in splice site a between the constitutive exons 12 and 13, termed a15, with or without an AAG triplett. These sites are thought to be important for the adhesive properties of NCAM. Therefore, these results suggest that modifications of NCAM may be important for adhesive interactions in normal and neoplastic endocrine cells.
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PMID:Neural cell adhesion molecules in rat endocrine tissues and tumor cells: distribution and molecular analysis. 844 Jan 82

The mouse ST8Sia II (mST8Sia II/STX) gene encodes a neural cell adhesion molecule-specific polysialic acid synthase whose expression is regulated during the developmental stages of mouse brain. To elucidate the molecular mechanism by which the expression is tissue-specifically and developmentally regulated, we isolated the complete genomic DNA and characterized the promoter of the gene for mST8Sia II. The gene encoding mST8Sia II was found to span about 80 kilobases and to be composed of six exons. Primer extension and S1 nuclease protection analyses revealed that the transcription started from 167 nucleotides upstream of the translational initiation site. Promoter analyses of the 5'-flanking region of the mST8Sia II gene using a luciferase gene reporter system revealed strong promoter activity in retinoic acid-induced differentiated P19 cells, which highly express the mST8Sia II gene. Deletion analyses demonstrated that the minimal promoter activity detected for the proximal region 325 base pairs upstream from the translational initiation codon (-158 to +167) could be modulated by various sequences within the 9. 5-kilobase 5'-flanking region. The minimal promoter was embedded in a GC-rich domain (74%, GC content), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. The positive regulatory region located between -159 and -659 contained two additional Sp1 binding motifs and a long pyrimidine-rich region. We also found that the minimal promoter region of the mST8Sia II gene was sufficient for expression of a reporter gene in mST8Sia II gene-expressing neural differentiated P19 cells but not in nonexpressing ones. Thus the TATA-less GC-rich minimal promoter region of mST8Sia II probably controls the cell type-specific expression of the mST8Sia II gene.
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PMID:Genomic structure and promoter activity of the mouse polysialic acid synthase gene (mST8Sia II). Brain-specific expression from a TATA-less GC-rich sequence. 893 67